Indonesian Journal of Biotechnology, June, 2009

Vol. 14, No. 1, pp. 1146-1154

Ethanolic Extract of Hedyotis corymbosa L. Increases Cytotoxic Activity of

Doxorubicin on MCF-7 Breast Cancer Cell
Sari Haryanti , Sendy Junedi and Edy Meiyanto
1

2 *)

1. Medical Plant and Traditional Medicine Research and Development Office, Tawangmangu,
Central Java, Indonesia
2. Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia

Abstract
Hedyotis corymbosa L. with ursolic acid as the main compound is one of the plants that has been used for
traditional medicine including to cure breast cancer disease. The aim of this research is to examine the cytotoxic
activity of rumput mutiara herb ethanolic extract (ERM) and its effect in combination with doxorubicin against MCF7 breast cancer cell line as cell model of doxorubicin resistance. Hedyotis corymbosa L. herb powder extraction was
done by maceration using ethanol 96% then the extract is detected for ursolic acid content. Cell viability assay of ERM,
doxorubicin and the combination of ERM and doxorubicin treatments were carried out by MTT assay to determine
IC and CI (Combination Index). Cell cycle distribution was determined by flowcytometry. Apoptosis assay was
performed by ethidum bromide-acridine orange DNA staining method. Investigation on Bcl-2 expression was
determined by immunocytochemistry method.
Thin Layer Chromatography of ERM had similar Rf with ursolic acid standard: 0,6. ERM and doxorubicin
inhibited cell growth against MCF-7 with IC of 77 g/mL and 349 nM (0,19 g/mL) respectively. Combination of
ERM and doxorubicin showed synergistic effect (CI 0.66-0.99). Combination of 25 g/mL ERM- 200 nM doxorubicin
induced apoptosis and decreased Bcl-2 expression but showed no cell accumulation on cell cycle. Doxorubicin
induced high cell accumulation in G2/M phase, but ERM at the concentration of 25 g/mL had a low effect in G1
phase, and ERM IC did not induce cell accumulation otherwise apoptosis. These results concluded that the apoptosis
mechanism of combination doxorubicin-ERM is mediated by cell cycle arrest and non cell cycle arrest. Therefore
ERM has a potential activity to be developed as co-chemotherapeutic agent.
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Keywords: Hedyotis corymbosa L., doxorubicin, co-chemotherapy, apoptosis , MCF-7

Introduction
Resistance to the chemotherapy is
common problem in cancer therapy. MCF-7
cell line characterized by p53 wildtype is one
of the breast cancer cell lines which is
resistance to doxorubicin due to over
expression of P-gp and Bcl-2 (Mealey et al.,
2001; Davis et al., 2003; Simstein, 2003). Co-

chemotherapy of doxorubicin with

chemopreventive agent is one alternative to
resolve the resistance in order to increase
efficacy and reduce toxicity of doxorubicin.
Rumput mutiara (Hedyotis corymbosa (L.)
Lamk, member of Rubiaceae family, at the
beginning was used for treating
appendicitis, peritonitis in China, then
nowadays it is developed as anticancer
(Dharmananda, 2007). The common
compound which is play apart in cytotoxic
activity of rumput mutiara is ursolic acid, a
pentacyclic triterpenoid. Ursolic acid was
known have anticancer activity through
inhibition of carcinogenesis, cancer

*Corresponding author : Dr. Edy Meiyanto, M.Si.,

Apt. Faculty of Pharmacy, Gadjah Mada University,
Sekip Utara, Yogyakarta 55281, Telp/Fax: (0274)
543120, E-mail : meiyan_e@yahoo.com
http://www.ccrc.farmasi.ugm.ac.id

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promotion and angiogenesis. Those

activities are mediated by the abilities of
ursolic acid to inhibit activation of nuclear
factor-kappaB (NF-eB). NF-eB is a protein
that regulating expression of some genes
which play a role in cancer formation
process; including antiapoptosis genes,
genes regulating molecule adhesion, and
genes regulating cell cycle (Shishodia et al.,
2003). Moreover, ursolic acid can induce
apoptosis by inhibiting cell cycle of MCF-7
breast cncer cells at G0/G1 phase and
increasing p53 expression (Zhang et al.,
2005). Therefore, a compound which is able
to inhibit NF-eB activation, increase p53
expression and inhibit cell cycle has the
therapeutic potency as anticancer agent.
This research is aimed to examine the
cytotoxic effect of rumput mutiara herb
ethanolic extract (ERM) and its combination
with doxorubicin on MCF-7 breast cancer
cell line. Moreover, analysis of apoptosis,
Bcl-2 expression and cell cycle are conducted
to understand the mechanism of those
cytotoxic effects. Finally, the result is going
to be the scientific evidence of Indonesian
herb usage in the effort to develop
Indonesian herb as combination agent
which can resolve resistance of doxorubicin
toward breast cancer cell.

Tatsuo Takeya (Nara Institute of Science and

Technology, Japan). Cells were cultured in
Dulbeccos modified Eagles medium
(DMEM) containing Fetal Bovine Serum
(FBS) 10.% (v/v) (Gibco), penicillinstreptomycin 1 % (v/v) (Gibco) and 0.25.%
trypsin-EDTA (Gibco) was used to detach
cells from flask and plate. For MTT assay,
use MTT [3-(4,5dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] (Sigma),
isopropanol acid (HCl 4N (Merck)isopropanol (1:100) (Merck). For apoptosis
assay, uses acridine orange (Sigma)ethidium bromide (Sigma). For
immunocytochemistry, antibody against
Bcl-2 (Cell signaling), universal detection kit
(streptavidin-HRP, biotinylated secondary
antibody, blocking serum, 3,3diaminobenzidin (DAB)) (Ultravision plus
detection system, Ref TP 125-HLX, Runcorn,
Cheshire, WA71PR, UK; Novostain
Universal Detection kit NCL-RTU,
Novocastra Lab Ltd., Newcastle NE12 8EW,
UK), mayers hematoxylin (Dako). For cell
cycle analysis usesTriton-X (Merck), RNAse,
propidium iodide (Sigma) in PBS.
Methods
Extraction of rumput mutiara herb
(Hedyotis corymbosa L.).
ERM was prepared by extracting the
dried herb with ethanol 96%. Then, the
menstrum was evaporated by rotary
evaporator and dried by freeze drying to get
the viscous extract.

Materials and Methods

Materials
Doxorubicin (Dox) was purchased from
Ebewe, PT. Ferron Par Pharmaceutical.
Hedyotis corymbosa L. herb was collected in
Solo, Central Java, and determined in Balai
Besar Litbang Tanaman Obat dan Obat
Tradisional, Tawangmangu, Central Java.
Materials for extraction and detection are
ethanol 96%, silika gel 60 F254 (Merck),
chloroform-aceton (9:1) (Merck), 10%
sulfuric acid and ursolic acid (Sigma). ERM
and Dox were dissolved in Dimethyl
Sulfoxide (DMSO) (Sigma). MCF-7 breast
cancer cell was kindly accepted from Prof.

Detection of ursolic acid

1% ERM and 0.1% ursolic acid in
methanol spotted on silica gel plate, then
eluted in chloroform-aceton (9:1). The plate
was dried and spried by 10% sulfuric acid
then heated at 110 C for 5 min and observed
under visible light.
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Assessment of cell viability

Cells were seeded at density of 5x10

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cells/ well in 96 well plate and incubated

with various concentration of ERM (50-200
mg/mL), Dox (115-1150 nM) and
combination of ERM (8, 20, 25, 40 mg/mL)Dox (4, 100, 135, 200 nM) for 48 h in incubator
37 C, 5% CO . Thereafter the medium was
changed and 100 L MTT 0.5 mg/mL was
added to incubate for 4 h in incubator 37 C.
The viable cells were directly proportional to
the production of formazan. Following
dissolution in 100 L isopropanol-HCl, the
absorbations were read at 595 nm with
ELISA reader (Bio-Rad microplate reader
Benchmark). IC value of ERM and Dox were
determined based on the equation of linier
regression of log concentration vs % cell
viability. Combination Index (CI) was
determined according to the equation
developed by Chou (Reynolds and Maurer,
2005). CI>1 shown antagonist, CI=1 shown
additive, and CI<1 shown synergy (Zhao et
al., 2004; Reynolds and Maurer, 2005).

cover slip were fixed with methanol and

rinse with PBS. Hidrogen peroxidase was
added to incubate for 10 minutes followed
with addition of prediluted blocking serum
for 10 minutes in room temperature.
Monoclonal antibody anti Bcl-2 was added
to incubate for 24 h in temperature 4 C.
Biotinylated secondary antibody was added
fro 10 min followed with addition of
streptavidin-peroxidase complex for 10 min.
Cells added with DAB for 10 minutes were
submerged in Mayers Haematoxylin for 3
min. The expression of Bcl-2 was observed
under light microscope (Nikon YS100). Bcl-2
expression was shown by brown color in
cytoplasm.

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Cell cycle analysis

Cell cycle analysis was performed with a
flowcytometer (FACS Calibur). 10 cells/
well were cultured in 6 wells plate and
treated with ERM (8, 25, 40 mg/mL), Dox
(40, 200 nM) and combination of ERM (8,
25mg/mL)-Dox (40, 200 nM) for 24 h.
Thereafter, the cells rinsed with PBS and
centrifuged at 2000 rpm for 3 min. The
attached cell was detached with 100 l
tripsin-EDTA for 3 min then added 1 mL of
DMEM and centrifuged at 2000 rpm for 30
sec. After that, cells rinsed with cold PBS and
centrifuged at 2000 rpm for 30 sec. Prior to
the samples being analyzed by the
flowytometry, propidium iodide (PI) stain
solution (7% Triton-X, 0.2% RNAse, 5%
propidium iodide) was added to the mixture
and it was incubated for 10 min in darkness
at temperature of 37 C. Data acquisition and
analysis were performed in the
flowcytometer with accompanying software
(Cell Quest). The percentage of hypodiploid
cells (sub G1 phase) over total cells was
calculated and represented as percent of
apoptosis.
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Apoptosis assay
Cells were seeded on cover slip at a
density of 3x10 cells/ well in 12 well plate
and incubated with ERM (25 mg/mL), Dox
(200 nM) and combination of ERM-Dox for
24 h at 37 C, 5% CO . Thereafter, the medium
was removed and rinse with PBS. 10 mL
acridin orange-ethidium bromide was
added to cover slip. Observation was
conducted by fluorescence microscope
(Zeiss MC 80). The color of apoptotic cell was
orange and there were nuclear
fragmentation and apoptotic body.
Otherwise, the color of normal cell was
green.
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Immunocytochemistry
Cells were seeded on cover slip at
density of 5x10 cells/ well and incubated
with ERM (25 mg/mL), Dox (200 nM) and
combination of ERM-Dox for 18 hours in
incubator 37 C, 5% CO . The medium was
removed and rinsed with PBS. Then, cells in
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Results and Discussion

Thin Layer Chromatography detection of

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ERM
TLC profile of ERM showed a pink spot
in visible light and the Rf (0.6) was similar to
the ursolic acid standard (Figure1). Rumput
mutiara contains pentacyclic triterpenoid of
ursolic acid and oleanolat acid which the
difference was only take place in the
location of one methyl group of E ring.
Therefore the separation of both
compounds was difficult in TLC even using
isocratic system. Based on that, the pink spot
in TLC profile of ERM was probably made
up from the combination of ursolic acid and
oleanolat acid.

probably caused by death cell but the

mechanism neither apoptosis nor necrosis
was not revealed yet.

Figure 2. Cell viability after ERM and doxorubicin

treatment on MCF-7 cell. The assessment were
conducted by incubating 5x10 cell with ERM (50-200
g/mL), Dox (115-1150 nM) for 48 hours. Treatment
ERM 125 g/mL (B) and Dox (506 nM) shown
morphological change compare with control cell (A).
Graphics of cell viability percentage vs ERM
concentration (D) and cell viability percentage vs Dox
concentration (E) shown linier correlation.
Magnification 100x. A.normal cell, B.morphologically
changed cell
3

Table 1. CI value of ERM and Dox combination on

MCF-7

Figure 1. TLC profile of ERM. Elution was used mobile

phase chloroform-aceton (9:1). Rumput mutiara herb
ethanolic extract is 1 and ursolic acid standard is 2. TLC
plate before (A) dan after (B) spraying with sulfuric
acid 10% in ethanol which was then heated in 110 C for
5 minutes was seen pink spot in visible light.

Combination treatment of ERM and Dox

gave synergistic effect at Dox concentration
200 nM and all ERM concentrations (8, 20, 25
and 40 g/mL) with CI values 0.66-0.99
(Table 1). These results revealed that ERM
has the ability to increase the sensitivity of
MCF-7 cell toward Dox. Dox concentration
below to IC did not show synergistic
effect instead it gave antagonistic effect. It
might be caused by dox and ERM
concentrations which were too low (1/10
1/3 IC ) therefore the combination effects
were not optimal.

Assessment of cell viability of rumput

mutiara herb ethanolic extract (ERM),
doxorubicin and its combination on MCF-7
Cell
ERM and doxorubicin inhibited cell
viability of MCF-7 cell with IC value 77
g/ml and 349 nM respectively (Figure 2).
According to the cell viability and cell
morphology, ERM and Dox treatment
showed linier correlation toward its
concentration. Morphological change was

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The effect of ERM, dox and its combination

on apoptosis
All cells in control cell gave green
fluorescence showing there was no death
cell. ERM and dox treatment caused some
cells gave orange and red fluorescence
which signed the increasing of cell
membrane permeability as the indicator of
apoptosis. The nuclear of several cells were
fragmented and formed apoptotic bodies.
The number of cells which were occurred
apoptosis was just few because the
concentration of ERM and dox were under
IC : ERM 25 g/mL (1/3 IC )and dox 200nM
(1/2 IC ). Furthermore the incubation period
was 24 hours, shorter than the period of
viability assessment. Combination
treatment of ERM and dox shown increasing
of apoptotic cells number compared with
single treatment of ERM and dox (Figure 3).
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Figure 3. Apoptotic induction after treatment of ERM,

dox and its combination on MCF-7 cell. 5x10 cells on
coverslips in 24 well plate were incubated for 24 hours
with ERM 25g/mL (B), dox 200 nM (C) and its
combination (D) then stained with acridine orangeethidium bromide and observed under fluorescence
microscope. Cell control was shown in A.
Magnification 100x, live MCF-7 cell, apoptotic cell
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The effect of ERM, dox and its combination

toward Bcl-2 expression on MCF-7 cell
Antiapoptotic protein, Bcl-2, is
expressed highly in MCF-7 cell and it is
correlated with low effect of chemotherapy
agent. Based on that, the exploration of ERM
and dox apoptotic mechanism either single
or combination were directed to inhibit Bcl-2
expression. The qualitative observation
shown the decreasing of Bcl-2 expression
after single treatment of ERM 25 g/mL, dox
200nM and both combination compared
with cell control (Figure 4). The decreasing
of Bcl-2 expression in combination treatment
was more than single treatment of ERM and
dox which was signed by low intensity of
brown color in cytoplasm compare with
single treatment. Therefore, each ERM and
dox was contributed to Bcl-2 expression in
combination treatment.

Figure 4. The effect of ERM, dox and its combination

toward Bcl-2 expression on MCF-7 cell. 5x10 cells were
seeded on coverslips in 24 well plate. Two coverslips
were as cell control and remains were treated with
ERM 25g/mL, dox 200 nM and its combination for 18
hours. Thereafter it was stained by using
immunocytochemistry with primer antibody anti-Bcl2 as mentioned in the method. (A) Cell control without
primery antibody Bcl-2, (B) cell control with with
vehicle treatment, (C) treatment of dox 200 nM, (D)
treatment of ERM 25 g/mL, (E) combination
treatment of dox and ERM (200 nM-25 g/mL).
Observation was done under light microscope, with
magnification 400x.
Cell expresses Bcl-2, cell does not express Bcl-2.
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The effect of ERM, dox and its combination

toward cell cycle modulation on MCF-7 cell
Distributions of MCF-7 cells in cell cycle
phases after single and combination
treatment were described in Table 2.
Table 2. Distribution of MCF-7 cell in cell phases after
treatment of ERM, dox and its combination.

Dox 40 nM dan 200 nM shown high cell

accumulation in G2/M phase but the cell
accumulation of dox 200 nM was less than
dox 40 nM because cells in sub G1 of dox 200
nM was more than dox 40 nM. The treatment
of ERM 25 g/mL caused low cell
accumulation in G1 phase but at
concentration IC (80 g/mL) there was no
accumulation in G1 phase. ERM 80 g/mL
shown increasing of sub G1 more than ERM
25 g/mL. Increasing of sub G1 cells was
showing the increasing of apoptotic cell and
this result was correlated with the result of
cytotoxic effect of dox and ERM.
The combination treatment of ERM-dox
8 g/mL-40 nM increased G1 and sub G1
accumulation and decreased G2/M
accumulation of dox single treatment (40
nM).This result showed the synergistic effect
of ERM and dox in cell cycle which is
dominated by dox. Therefore, G2/M phase
was still high. The cell cycle profile of ERMdox 25 g/mL-200 nM was similar to cell
control except sub G1 (Figure 5.H) so the
synergistic effect of its combination was
mediated by apoptosis. The result above
revealed that the synergistic effect was not
only mediated by cell cycle accumulation
but also apoptosis
ERM inhibit the viability of MCF-7 cell
with low IC (77 mg/mL, less than 100
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Figure 5. Cell cycle analysis of MCF-7 after treatment

of ERM, dox and its combination. 10 cells were seeded
in 6 well plate and incubated with ERM (8, 25, 80
g/mL), dox (40, 200 nM) and combination ERM-dox
(8 g/mL-40nM; 25 g/mL-200 nM) for 24 hours. Cells
were harvested then added with PI reagent and
analyzed with flowcytometer. (A) cell control, (B) ERM 8
g/mL, (C) ERM 25 g/mL, (D) ERM 80 g/mL, (E)
dox 40 nM, (F) dox 200 nM, (G) ERM-dox 8 g/mL40nM, ERM-dox 25 g/mL-200 nM.
4

mg/mL) so it is potential to develop as

alternative chemotherapy agent. TLC profile
shown pink spot which Rf was similar with
Rf of standard ursolic acid. The spot
contained of ursolic acid and oleanolat acid
that difficult to separate because of similar
structure. In MCF-7 cell, ursolic acid has IC
4,7 g/mL while oleanolat acid has lower
activity than it. Until concentration 20
g/mL, oleanolat acid only inhibited 30% of
MCF-7 cell viability (Chen et al., 2005). Based
on that research, ursolic acid was more
contributed to cytotoxic activity than
oleanolat acid in MCF-7 cell.
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MCF-7 cell has lowsensitivity to dox

showed by the high IC , that was 349 nM.
That value was much bigger than IC dox on
T47D breast cancer cell, that was 43 nM
(Jenie and Meiyanto, 2007). Viability
assessment of combination treatment by
using high concentration of dox (200 nM)
and various concentration of ERM (8, 20, 25
dan 40 g/mL) gave low CI categorized in
synergistic effect and the result was
correlated to apoptosis assay. Combination
treatment of dox 200 nM and ERM 25 g/mL
increased apoptosis incident of dox and
ERM single treatment. That synergistic effect
can be used as the basic of cochemotherapeutic development.
The exploration of apoptosis
mechanism was correlated with expression
of Bcl-2 regulator protein. The expression of
Bcl-2 was decreasing after treatment of dox,
ERM and its combination. One protein
controlling Bcl-2 expression is NF-eB, a
transcription factor (Aggarwal et al, 2006).
NF-eB is expressed highly in malignancy
process of breast cancer (Shehata, 2005).
Ursolic acid has the ability to decrease
activation of NF-eB through inhibit IeBa
kinase and phosphorilation of p65 which
was correlated with the decreasing of cyclin
D, COX-2, and MMP 9 expression.
Therefore, the decreasing of Bcl-2expression
which then inducing apoptosis in ERM
treatment was probably mediated by ursolic
acid. ERM which inhibited Bcl-2 expression
can also increase FasL expression
(Srivastava, 1999) while dox can induce Fas
receptor expression (Yamaoka et al., 2000).
Both mechanism probably produce
synergistic effect in combination treatment
of ERM and dox, mediated by intrinsic and
extrinsic pathway of apoptosis.
Analysis of cell cycle can explain further
about synergistic mechanism of its
combination treatment. Dox induce cell
accumulation in G2/M phase at all range of

concentrations while ERM induce cell

accumulation in G1 phase at concentration
25 mg/mL (1/3 IC ). In higher concentration
of ERM, 80 mg/mL (IC ) percentage of
apoptotic cell increased and it was probably
happened because p53 was expressed highly
by ERM induction. Whereas, in low
concentration of ERM (25 mg/mL), p53
expression was probably relative low so cell
cycle arrest was more than apoptosis. Cell
accumulation in G2/M phase after treated
with dox was probably caused by increasing
of p53 dan p21 expression through Ras
pathway. Then it was followed by
upregulation of cyclin D3 and E, and
downregulation of p16. Therefore, cell
passed G1 checkpoint yet detained in G2/M
phase, because there was no upregulation of
cyclin B. Different with dox, the increasing of
p53 expression induced by ERM probably
did not cause upregulation of p21 so there
were downregulation of cyclin D3 and E,
and upregulation of p16. Thereafter cell was
detained in G1 checkpoint (Mansilla et al,
2003; Zhang et al, 2005).
Combination treatment of dox-ERM 40
nM- 8 mg/mL shown synergistic effect with
cell accumulation in G2/M phase. The
G2/M cell accumulation which was like dox
single treatment was happened because the
effect of ERM low concentration (8 mg/mL)
was lower than dox. The synergistic effect
was also happened in dox-ERM 200 nM- 25
mg/mL with increasing of apoptosis but no
inhibition of cell cycle. In combination of
dox-ERM 200 nM- 25 mg/mL, either ERM or
dox gave effect to cell cycle by increasing p53
expression. Therefore p53 expression of doxERM 200 nM- 25 mg/mL was much higher
than single treatment and it made increasing
of apoptosis incident.
Apoptotic mechanism of combination
treatment dox-ERM in MCF-7 cell was
mediated by cell cycle arrest and non cell
cycle arrest. The mechanism of non cell cycle

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WAF1

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arrest was probably mediated by decreasing

of NF-eB activation and increasing of FasL,
Fas and p53 expression. The ability of ERM
to increase cytotoxic activity of doxorubicin
mediated by various mechanisms of
apoptosis make ERM has the potency to
develop as co-chemotherapeutic agent with
doxorubicin in breast cancer. In addition,
rumput mutiara (Hedyotis corymbosa L.)
grow easily and not used widely yet so it is
more potential to develop further.

procumbens (Lour.) (Merr.) terhadap

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Acknowledgment
We thanks to Prof. Tatsuo Takeya (Nara
Institute of Science and Technology, Japan) for
the giving of MCF-7 cell (human mammary
carcinoma cell).
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